Sterilization of female mosquitoes with a hormone derived from male mosquitoes



3,450,816 STERILIZATION F FEMALE MOSQUITOES WITH A HORMONE DERIVED FROMMALE MOSQUITUES George B. Craig, Jr., and Morton Fuchs, South Bend,

Ind., assignors t0 the United States of America as represented by theSecretary, Department of Health, Education, and Welfare No Drawing.Filed Nov. 28, 1967, Ser. No. 686,336 Int. Cl. Allin 9/00, 23/00; C07q17/00 US. Cl. 424-95 4 Claims ABSTRACT OF THE DISCLOSURE A method ofsterilizing female mosquitoes with a substance extracted from theaccessory glands of male mosquitoes, the method of extracting andpurifying the substance, and the purified material, named matrone.Matrone is obtained from the male terminalia by extraction with salinesolution or, alternatively, by extracting whole male bodies with organicsolvents, followed by extraction of matrone with saline solution.Matrone can be lyophilized and stored. Female mosquitoes to whichmatrone has been administered by injection or feeding are sterilized forlife.

This invention relates to a composition and a method for the control ofinsects, and more particularly for the control of mosquitoes.

It is well known that various insects such as mosquitoes, flies, ticksand mites, to name just a few, are carriers of diseases which haveplagued mankind through the ages. The earliest, and most successfulmethods of disease prevention through insect control have utilizedinsecticides of one type or another. The disadvantages accompanying theuse of insecticides are well known due to the increased publicity inthat area in recent years. It has been found that many of the commonlyused insecticides have an extremely toxic effect to Wild life, tovegetation, and to humans. The toxicity of several such chemicals hasonly recently been demonstrated since it involves a cumulative effectand is not immediately obvious. The important, and tragic consequencesof these various chemicals has taken on new importance with thediscovery that ecological imbalances have resulted. The persistent andenvironmental contamination of many extremely toxic insecticides hasalso become a cause for public alarm. Another, extremely importantdiscovery, has been that many insects gradually become resistant tospecific insecticides, thereby creating the need for using progressivelystronger and more dangerous chemicals. The danger accompanying the useof these compounds cannot be underestimated.

Over the last decade considerable research has been performed resultingin various experimental methods of sterilizing specific insects so that,by a natural process of attrition, the insect population would dwindle.Notable success has been achieved in this area using such sterilizationmethods as radiation as well as the use of chemicals and hormones. Thereare obvious drawbacks to those methods, not the least of which isfinding methods of treating great numbers of insects.

It is, therefore, an object of this invention to provide a compositionand a method for the control of insects by sterilization.

It is another, important, object of this invention to provide a compoundand a method suitable for widespread dissemination for the control ofinsects by sterilization.

It is yet another object of this invention to provide a composition andmethod which will effectively sterilize tates atent 1female mosquitoes,thereby depleting the mosquito popuation.

It is known that insemination stimulates egg deposition in Aedesaegypti. It was shown that the mechanics of mating were not responsiblefor the stimulus since males which had mated repeatedly and hence hadtheir supply of seminal fluid depleted, failed to stimulate females tooviposit, even after frequent copulation. Later, it was hypothesizedthat a stimulant substance is produced by the male accessory gland whichwas responsible for the stimulation of oviposition. In order to provethis hypothesis, experiments were performed in the implantation of maleaccessory glands into the thorax of virgin females of two species ofAedes, A. aegypti and A. albapictusx In this work the reproductive tractwas extracted from virgin males and the glands were removed therefrom.The glands were then inserted into the thorax of the females andcompared with various controls. There was a striking increase in theoviposition of the treated females.

Later work in the field of genetics produced remarkable results. Whenseveral males of Aedes aegypti, each with a. different genetic marker,were placed with a single female, all progeny came from the first malethat copulated with the female. This was despite the fact that repeatedcopulation was observed. Thus, the widely held assumption that a singlefemale can be inseminated by several males was disproved. A further,most unexpected, observation was that although copulation may take placemany times, a female mated only once is refractory to subsequentinsemination for life. Multiple insemination can take place only whenseveral males copulate with a female in a short period of time.

The agent which stimulates this reaction in females was found to comefrom male accessory glands and to be passed to the female in seminalfluid. As will be described in more detail hereinbelow, implantations ofmale accessory glands into the thorax of females of A. aegypti was foundto inhibit insemination when the females were then exposed to virginmales. This sterilizing effect lasts for the entire life of the female.Even though the treated female oviposited, none of the eggs hatched. Themechanism of action of the accessory gland substance in the female hasnot yet been determined. The sound-in-fiight of females with glandimplants is fully attractive to males, but such females sometimes avoidcopulation by lowering the abdomen to the surface or otherwisepreventing the male from assuming the coital position. Males placed withonce-mated females often go through the preliminary steps of coitus butfail to ejaculate. In normal coitus in A. aegypti, male paraprocts areinserted between the genital lips, opening them and facilitatinginsertion of the aedeagus. Perhaps the accessory gland stimulates thefemale to hold the vaginal lips closed. If so, the lips might bebreeched occasionally by the mechanical pressure of forced mating butnot by the male in the normal act of mating. Certainly, force-mating ismuch more readily accomplished with virgins than with once-matedfemales. It has been found that males, force-mated to females with glandimplants, seemed to have difiiculty in inserting the aedeagus andexternal ejaculation was fairly frequent. On the other hand, it has beensuggested that semen is introduced into the female in second matings butis subsequently expelled due to cuticular changes in the female genitalattrium induced by accessory gland substance.

The effect of male accessory substance was similar in Culex pipiens,Anopheles quadrimaculatus, Aedes aegypti, A. albopictus, A. atropalpus,A. mascarensis, A. poly- Leahy, M. G., and Craig, G. B., Mosquito News,25, 448 (1965).

nesiensis, A. scutellaris, A. sierl'ensis, A. togoi, A. triseriatus, andA. vittatus. Both homologous and heterologous transplants were made,with results that differed only in degree. It was even found that theglands of Drosophila melanogaster afforded protection in A. aegypti,even though multiple insemination is common in D. melanogaster.

Further, it was determined that the male accessory gland substance,which has been named matrone, could be extracted and purified foradministration to females. As will be described in detail below, matronecould be extracted from either the terminalia of the males, or from thecomplete body and administered in one of several ways. Each maleapparently contains enough active matrone to sterilize at least 64females. In experimental use, a solution of matrone injected into thethorax of a virgin female was found to sterilize the female for life. Asa more practical method of dissemination of matrone, it was found thatfemales fed on this substance in sugar solution are also sterilized. Itis, therefore, apparent that this substance may be fed to femalemosquitoes, thereby sterilizing them for life. Alternatively, matronecould be applied in the food of larvae or it could be applied, as byspraying, on surfaces where newly-emerged mosquitoes may rest.

The invention is further illustrated by the following examples, althoughit is not intended to be limited thereto.

EXAMPLE I To establish that the agent which stimulates monogamy infemales comes from male accessory glands and is passed to the female inseminal fluid various male tissues were dissected in saline solution andimplanted in the thorax of virgin females. The tissues used were maleaccessory gland, male gut, and testis. Further controls were used withno tissue, but with a sham-operation. After a 24-hour period ofrecovery, the females were placed in a cage with a surplus of virginmales, where they remained an additional 24 hours. The females were thendissected and examined for sperm in the spermathecae (the sole storageorgan for sperm in mosquitoes). As can be seen in Table I, at least 85%of the control females were inseminated, compared to for females withimplanted male accessory glands.

TABLE I.-EFFECT OF TRANSPLANTATION OF VARIOUS TISSUES INTO THE THORAX OFVIRGIN FEMALES ON SUBSEQUENT INSEMINATION IN AEDES AEGYPTI (ROCK STRAIN)Females Time after implant (females placed with N o. Virgins No.Insemmated males) (hours) Implanted Examined (percent) NO TISSUE(SHAM-OPERATED) TESTIS MALE GUT MALE GLAND *Treated females kept. with asurplus of males for 24 hours and then dissected and examined forpresence of sperm in spermathecae.

Apparently, several hours are required before the sterilizing effect ofaccessory gland material is accomplished. When females with glandimplants were placed with males immediately after operation, 26% wereinseminated. 5% of females that were allowed 4 hours for recovery wereinseminated, while those given longer periods had complete protectionfrom. insemination. The sterilizing effect lasts for the entire life ofthe female.

EXAMPLE II In this experiment, glands were implanted in females lessthan one hour old. Each week for ten weeks, these females received ablood meal and were placed with a new batch of males one to two weeksold. At the end of ten weeks, when 188 females with gland implants weredissected, none was inseminated. In the control with gut implants, 155to 157 females were inseminated. Some females were isolated individuallywith 5 males. Repeated blood meals were provided, and each female wasplaced with 5 fresh males after each oviposition. Each of 5 females withgland implants produced 6 separate egg batches, with total eggproduction of 357, 314, 292, 275 and 258. None of these eggs hatched.Five females with gut implants also produced six separate batches,giving 327, 302, 30-0, 249 and 246 eggs. More than of the eggs from eachbatch were hatchable.

EXAMPLE III The effect of male accessory gland substance was similar inCulex pipiens, Anopheles quadrimaculatus, and ten species of Aedes (A.aegypti, A. albopictus, A. atropalpus, A. mascarensis, A. polynesiensis,A. scutellaris, A. sierrensis, A togoi, A, triseriatus and A.vittatius). After 24 hours, operated females were exposed to homologousmales for 96 hours. Insemination in the controls with gut implantsvaried from 70% for Anopheles to 100% for C. pipiens. No inseminationoccurred in any female with gland implants among the 12 species tested.These results seem to indicate that female monogamy is widespread inmosquitoes.

Heterologous transplants between species gave some protection frominsemination, but the effect was less pronounced. For example, 40females of A. aegypti gave the indicated percentage of insemination whenimplanted with male glands of the following species:

A. atropalpus 15 A. triseriatus 33 Culex pipiens 50 A. scutellaris 63Drosophila melanogaster 30 With females of A. triseriatus, glands of A.aegypti gave 8% of those of A. atropalpus gave 10%. In females of Culexpipiens, glands of A. albopictus gave 10%, and those of A. aegypti gave65%. Thus, there is no apparent correlation between phylogeneticposition and amount of protection. Even glands of Drosophila gaveprotection in A. aegypti, although multiple insemination is common in D.melanogaster. Other insects may serve as a source of material which willprevent insemination in mosquitoes. There is even evidence that femalemonogomy exists in the housefly. In this species, females lose sexualreceptivity after injection or implantation of material from the maleejaculatory duct.

EXAMPLE IV While identification of the active principle in maleaccessory glands (matrone) of A. aegypti has not been completed,extraction and purification have been performed. The following methodhas been used for the preparation of stock solutions:

(1) The terminalia and abdominal segment VIII of 250 males, preferablyvirgin and over 4 days old, was placed in 1 ml. of saline;

(2) The mixture was sonicated for 1 minute;

(3) The mixture was then centrifuged for 30 minutes at 4,000 g. and theresidue was discarded; and,

(4) The supernatant was frozen for storage.

The assay procedures to determine activity in A. aegypti were asfollows:

(1) 20 virgin females were anesthetized;

(2) 1 ,ul. of solution was injected into the thorax of each female;

(3) 24 hours were allowed for recovery, and then the females were placedwith 40 males; and

(4) 48 hours after injection, the females were dissected and thespermathecae were examined for insemination.

Control females injected with saline generally show 85 to 95%insemination after 24 hours with males.

Two-fold serial dilutions were used to determine activity of stocksolution by titration. The results can be seen in Table II.

TABLE II.ASSAY OF MALE ACCESSORY GLAND ACTIV- IIY IN AEDES AEGYPTI BYINJECTION F FEMALES WITH SERIAL DILUTIONS OF VARIOUS GLAND PREP-ARATIONS.

Females Inseminated From Treated Preparation *Stock solution from 250male terminalia in 1 ml. of saline. Injection of 1 #1. gave a doseequivalent to one-fourth of a pair of male glands.

T'Iwenty females injected for each dilution, placed with males 24 hourslater, and examined for insemination 48 hours after injection.

Glands taken from males fed on slices of canned apple gave slightly moreactivity than those from males fed on dry sugar cubes. The first columnof results in Table II is typical of many other assays of stocksolution. Each male apparently contains enough matrone to sterilize atleast 64 females. The initial stock solution contained glands from 250males in 1 ml. of saline. Each female received 1 1., a dose equivalentto one-half gland or one-fourth of a pair of glands. Thus, the glandmaterial from one male was distributed among four females. The stocksolution could be diluted 1 6 times and yet retain enough activity forcomplete prevention of insemination.

The stock solution of matrone from A. aegypti is clear and colorless,and possesses the following characteristics: (1) It is stable in cold.Activity is retained indefinitely while the solution is frozen at 20 C.and is reduced only slightly by repeated freezing and thawing. It can bestored frozen or at C.

(2) It is unstable in heat. It was destroyed by 5 minutes at 50 C., butwas unaffected by 40 C.

(3) It is pH-sensitive. It is denatured above or below 4. 4

(4) It is lyophilizable. The lyophilized material can be stored dry atroom temperature indefinitely.

(5) It is non-dialyza'ble. (6) It is readily precipitated with 50%ammonium sulfate solution.

(7) It is destroyed by amyl alcohol and chloroform. (8) It is notprecipiated, nor affected by manganous chloride.

(9) The molecular weight is estimated to be about 10,000 as measuredfrom Sephadex columns.

These characteristics are consistent with the hypothesis that thesubstance is a protein, and probably a globulin protein.

EXAMPLE V The following procedure was used to extract matrone from wholebodies of male A. acgypti.

Frozen whole body, five day old adult males were lyophilized to dryness,resulting in a weight reduction of about 40-50%. Fine glass beads wereadded in an amount equal to the weight of the water lost. Approximatelyfive volumes of acetone at C. was added and the mixture was homogenizedin the cold for five minutes.

The homogenate was filtered, the solid material was resuspended in fivevolumes of n-butanol at 15 C., and the homogenization was repeated. Thematerial was again filtered and the solid material was washed withapproximately 100 ml. of acetone at 15 C. on the filter paper. Thegrayish-brown powder remaining on the filter paper was collected forfurther processing. This material could be stored in a freezer.

The powder was next suspended in 5-10 volumes of A. aegyptiphysiological saline solution and homogenized for 15 minutes in thecold. The homogenate was centrifuged at 48,000 g. for 20-30 minutes andthe precipitate was discarded. It should be noted that these stepsshould be performed in the cold.

To the cold supernatant, 1/50 volume of 1.0 N manganous chloride wasslowly added to remove the nucleic acids as a precipitate. After 4-12hours the material was again centrifuged as before and the precipitatewas discard. Solid ammonium sulfate was dissolved in the supernatant tomake a 60% saturated solution. The solution was kept cold for 4-12 hourswith stirring and then centrifuged as before and the supernatant wasdiscarded.

The precipitate was redissolved in cold physiological saline anddialyzed overnight in the cold against a large volume (at least 1-2liters) of saline. The remaining concentrated solution was assayed foractivity as described hereinbelow.

' EXAMPLE VI The procedure of Example V was repeated but the finalconcentrated solution was lyophilized to a dry powder and stored at roomtemperature.

EXAMPLE VII TABLE III Percent inseminated at pH (N=20 females) Dilution1.5 3.0 4.3 4.9 5.2 6.8 8.7 9.5 10.2 12.0

males.

EXAMPLE VIII In order to determine the feasibility of administration ofmatrone in food, the following procedure was followed:

(1) Virgin females were held without water for 24 hours;

(2) A cotton pad soaked with matrone (250 males per ml.) and 10% sucrosewas provided for 24 hours;

(3) The females were then placed with males;

(4) A control was run under the same conditions using sucrose alone.

The results were as follows: of 20 females tested, 2 were inseminated;of 20 control females, 20 were inseminated.

Thus, it can be seen that matrone can be administered in food toeffectively sterilize female mosquitoes.

Since it has been noted that A. aegypti cannot be inseminated until atleast 48-50 hours after adult emergence, and ample feeding takes placeduring this period, simple and inexpensive methods of distributionshould prove effective.

It should be understood that saline solution, as used herein refers toAedes aegypti physiologoical saline solution.

What is claimed is:

1. A method of purifying matrone comprising the steps of:

(a) lyophilizing the whole bodies of male mosquitoes to dryness;

(b) extracting said whole bodies with acetone at about 15 C. to obtain afirst residue of impure matrone;

(c) extracting said first residue with n-butanol at about -l5 C. toobtain a second residue of impure matrone;

(d) extracting said second residue with Aedes aegypti physiologicalsaline solution in the cold to obtain a saline solution of matrone;

(e) adding 5 th volume of 1.0 N manganous chloride to remove impuritiesby precipitation, and separating the resulting precipitate from theresulting supernatant;

(f) adding solid ammonium sulfate to said supernatant to make a 60%saturated solution to remove matrone by precipitation and separating theresulting precipitated matrone from the resulting supernatant; and,

(g) redissolving said matrone in cold Aedes uegypti physiological salinesolution and concentrating the resulting solution by dialysis.

2. The product produced by the method of claim 1.

3. A method in accordance with claim 1, further comprising the steps of:

(h) removing said saline solution of matrone of step (d) from theresulting precipitate by centrifuging; and,

(i) performing said separations in steps (e) and (f) by centrifuging.

4. A method according to claim 3, further comprising recovering purifiedmatrone from the concentrated matrone solution of step (g) bylyophilization.

References Cited Journal of Economic Entomology, vol. 46, No. 4 (1953)pp. 624-626.

Mosquito News, v01. 25, No. 4 (1965) pp. 448-452.

ALBERT T. MEYERS, Primary Examiner.

F. E. WADDELL, Assistant Examiner.

US. Cl. X.R. 424l05

